The invention provides isolated nucleic acid and amino acid sequences of Xenopus CENP-E (XCENP-E), antibodies to XCENP-E, methods of screening for CENP-E modulators using biologically active CENP-E, and kits for screening for CENP-E modulators.
Segregation of genetic material during mitosis is mediated by the microtubules of the mitotic spindle (see, e.g., McIntosh, in Microtubules, pp. 413-434 (Hyams and Lloyd, eds., 1994). During mitosis, chromosomes are dynamically attached to spindle microtubules via the kinetochore, which is a structure located at the centromere of the chromosome. Kinetochores are involved in coordinating chromosome movement via microtubule assembly and disassembly. The kinetochore and its component proteins thus play an important role in the dynamics of mitosis.
Spindle microtubules have a defined polarity, with their slow-growing, xe2x80x9cminusxe2x80x9d ends anchored at or near the spindle pole, and their dynamic, fast-growing xe2x80x9cplusxe2x80x9d ends interacting with chromosomes (McIntosh, et al., J. Cell Biol. 98:525-533 (1984)). During prometaphase, chromosomes establish interactions with the fast-growing plus ends of microtubules via the kinetochore. Chromosomes then undergo a series of microtubule-dependent movements, culminating in alignment at the metaphase plate, equidistant from the two spindle poles. This process is called xe2x80x9ccongression.xe2x80x9d However, the molecular mechanisms underlying chromosome congression are poorly understood (see, e.g., Rieder, et al., J. Cell Biol. 124:223-33 (1994)). A major question has been whether any kinetochore-associated microtubule motors play an important role in congression.
The two predominant and opposing forces are currently thought to be responsible for chromosome movement during congression: (1) an anti-poleward polar force associated with regions of high microtubule density near the spindle poles, and (2) a poleward force generated at the kinetochore (Khodjakov, et al., J. Cell Biol. 135:315-327 (1996); Waters, et al., J. Cell Sci. 109:2823-2831 (1996); reviewed in Rieder, et al., Int. Rev. Cytol. 79:1-57 (1982); Mitchison, et al., Annu. Rev. Cell Biol. 4:527-49 (1988); Rieder, et al., J. Cell Biol. 124:223-33 (1994)).
Studies in vitro have demonstrated the presence of both plus and minus end-directed microtubule motor activities on kinetochores that may be responsible for these chromosome movements (Mitchison, et al., J. Cell Biol. 101:766-77 (1985); Hyman, et al., Nature 351:206-211 (1991)). The outstanding issue, however, has been the identity of the molecules at the kinetochore which act as motors and generate the force for chromosome movement.
In general, both genetic and biochemical approaches have demonstrated crucial roles for microtubule motors in spindle assembly, spindle pole separation, and regulation of spindle microtubule dynamics. These motors include Eg5, CHO1/MKlp1, ncd, cut7, bimC, CIN8, KIP1, KAR3, Xklp2, XKCM1, and XCTK2 (Sawin, et al., Nature 359:540-543 (1992); Blangy, et al., Cell 83:1159-1169 (1995); Sawin, et al., J. Cell Biol. 112:925-940 (1991); Nislow, et al., J. Cell Biol. 111:511-522 (1990); Endow, et al., J. Cell Sci. 107:859-867 (1994); Hagan, et al., Nature 347:563-566 (1990); Hagan, et al., Nature 356:74-76 (1992); Enos, et al., Cell 60:1019-1027 (1990); Hoyt, et al., J. Cell Biol. 118:109-120; Roof, et al., J. Cell Biol. 118:95-108 (1992); Saunders, et al., Cell 70:451-458 (1992), Boleti, et al., J. Cell. Biol. 125:1303-1312; Walczak, et al., Cell 84:37-47 (1996); Walczak, et al., J. Cell Biol. 136:859-70 (1997)). Two kinesin superfamily members, Xenopus Xklp1 and Drosophila nod localize to chromosome arms. With the exception of these two chromatin-associated motors, which are thought to mediate polar ejection forces, none of these other proteins have been implicated directly in congression or in chromosome movement during other phases of mitosis (Theurkauf, et al., J. Cell Biol. 116:1167-1180 (1992); Afshar, et al., Cell 81:129, Cell 81:128-138 (1995); Vernos, et al., Trends in Cell Biol. 5:297-301 (1995)).
A candidate for powering chromosome movement in mitosis is centromere-associated protein-E (CENP-E), a member of the kinesin superfamily of microtubule motor proteins. Human CENP-E has been cloned and is an integral component of the kinetochore (Yen, et al., Nature 359:536-539 (1992); Yao, et al., The microtubule motor CENP-E is an integral component of kinetochore corona fibers that link centromeres to spindle microtubules (manuscript)). CENP-E localizes to kinetochores throughout all phases of mitotic chromosome movement (early prometaphase through anaphase A) (Yen, et al., Nature 359:536-539 (1992); Brown, et al., J. Cell. Biol. 125:1303-1312 (1994); Lombillo, et al., J. Cell Biol. 128:107-115 (1995)).
Previous efforts have suggested a role for CENP-E in mitosis. Microinjection of a monoclonal antibody directed against CENP-E into cultured human cells delays anaphase onset (Yen, et al., EMBO J. 10:1245-1254 (1991)). Anti-CENP-E antibody injection into maturing mouse oocytes induces arrest at the first reductional division of meiosis (Duesbery, et al., Proc. Natl. Acad. Sci. USA (in press, 1997)). Antibodies against CENP-E block microtubule depolymerization-dependent minus end-directed movement of purified chromosomes in vitro (Lombillo, et al., J. Cell Biol. 128:107-115 (1995)).
However, these experiments have not demonstrated the precise role of CENP-E in mitosis, nor have they shown the activity of CENP-E, in particular any motor activity. Recently, CENP-E was reported to be associated with minus end-directed microtubule motor activity, raising the possibility that CENP-E might be responsible for poleward kinetochore movements (Thrower, et al., EMBO J. 14:918-926 (1995)). However, biologically active CENP-E has never been isolated, neither from naturally occurring nor recombinant sources.
The present invention provides for the first time biologically active CENP-E and surprisingly demonstrates, contrary to previous reports, that CENP-E is a motor that powers chromosome movement toward microtubule plus ends. Using immunodepletion and antibody addition to Xenopus egg extracts, the present invention further demonstrates that CENP-E plays an essential role in congression. The present invention also provides for the first time the nucleotide and amino acid sequence of isolated Xenopus CENP-E.
In one aspect, the invention provides an isolated, biologically active CENP-E protein, wherein the CENP-E protein has the following properties: (i) at least one activity selected from the group consisting of plus end-directed microtubule motor activity, ATPase activity, and microtubule binding activity; and (ii) the ability to specifically bind to polyclonal antibodies generated against CENP-E. In one embodiment, the CENP-E protein has an average molecular weight of about 300-350 kDa.
In one embodiment, the CENP-E protein has an amino acid sequence having at least 34%, or alternatively at least 45%, or alternatively at least 55% sequence identity with a XCENP-E motor domain of SEQ ID NO:1. Alternatively, CENP-E has at least 60%, 65% or 70% sequence identity with a XCENP-E motor domain of SEQ ID NO:1. In an alternative embodiment, the CENP-E has 70%, or alternatively 75%, or alternatively 80%, or alternatively 85%, or alternatively 90% or alternatively 95% amino acid sequence identity to a Xenopus CENP-E core motor domain as measured using a sequence comparison algorithm. In an alternative embodiment, the CENP-E protein has an amino acid sequence of SEQ ID NO:1.
In another embodiment provided herein, the CENP-E protein is encoded by a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO:2. In another aspect of the present invention, the CENP-E protein is encoded by a nucleic acid which hybridizes under high stringency to a nucleic acid having a sequence complementary to that of SEQ ID NO:2.
In one embodiment, the CENP-E protein is from a human. In alternative embodiments provided herein, the CENP-E protein is from fungus, insects, or plants.
In an alternative embodiment provided herein, the CENP-E protein specifically binds to antibodies generated against Xenopus CENP-E (XCENP-E). In this embodiment, the CENP-E protein has an amino acid sequence having greater than 70%, or alternatively 75% sequence identity with a XCENP-E motor domain of SEQ ID NO:1. In another embodiment, the CENP-E protein has an amino acid sequence of a XCENP-E motor domain of SEQ ID NO:1.
In the embodiments wherein the CENP-E is biologically active as described herein, the amino acid sequence can have 74% or less sequence identity with the motor domain of SEQ ID NO:1.
Also provided herein is an isolated nucleic acid sequence encoding a CENP-E gene product, said sequence encoding a protein having a core motor domain that has greater than 70% or alternatively 75% amino acid sequence identity to a Xenopus CENP-E (XCENP-E) core motor domain as measured using a sequence comparison algorithm, and specifically binding to antibodies raised against CENP-E. In one embodiment, the sequence has a nucleotide sequence of SEQ ID NO:2. The sequence comparison algorithm can be PILEUP.
In another aspect of the invention, an antibody which specifically binds to CENP-E is provided.
Also provided herein is a method for identifying a candidate agent as a compound which modulates CENP-E activity. The method comprising the steps of determining CENP-E activity in the presence of a candidate agent at a control concentration. The CENP-E activity is selected from the group consisting of plus end-directed microtubule motor activity, ATPase activity and microtubule binding activity. The method further comprises the steps of determining said CENP-E activity in the presence the candidate agent at a test concentration, wherein a change in activity between the test concentration and the control concentration of said candidate agent indicates the identification of a compound which modulates CENP-E activity. The method can further comprise the step of isolating biologically active CENP-E from a cell sample.
The compound to be identified can be a lead therapeutic, bioagricultural compound or diagnostic. Preferably the compound is an antibody which specifically binds CENP-E. In one embodiment the method further comprises the step of modifying the antibody to be a humanized antibody. In one embodiment, the method is performed in a plurality such that many candidate agents are screened simultaneously.
The invention also includes kits for screening for modulators of CENP-E. The kit includes a container holding biologically active CENP-E and instructions for assaying for CENP-E activity, wherein the CENP-E activity is plus end-directed microtubule motor activity or ATPase activity.
The invention also provides a method of producing a biologically active CENP-E polypeptide. The method includes the steps of transforming a cell with a vector comprising the nucleic acid sequence encoding the motor domain of CENP-E; expressing said nucleic acid to produce a gene product; purifying said gene product; and identifying ATPase activity or plus-end directed microtubule activity of said gene product.
In another aspect of the invention, a method of moving microtubules in a plus ended direction is provided wherein microtubules are contacted with biologically active CENP-E.
In one embodiment the CENP-E is provided in gene form to a cell comprising microtubules.